The field of the present invention is the area of medical treatment and diagnosis using molecular technology. In particular, the present invention utilizes gene therapy and drug induced gene expression to ameliorate the physical condition of muscular dystrophy patients, especially those lacking dystrophin or lacking dystrophin and utrophin or those with lower than normal levels of α7 integrin, and in another aspect, this invention relates to the use of nucleic acid probes or primers or immunological probes for detecting the reduction of or lack of expression of the α7β1 integrin in scapuloperoneal muscular dystrophy (SPMD) as well as to the use of assays to identify compounds which induce increased expression via α7β1 integrin transcriptional regulatory sequences.
Scapuloperoneal (SP) muscular dystrophy, is one of a heterogeneous group of scapuloperoneal syndrome affecting the muscles of the shoulder girdle and peroneal. SP syndromes were formerly grouped as one genetic disease, but clinical analysis and genetic mapping have revealed that this syndrome includes at least two distinct diseases with different underlying genetic defects. SPMD is an autosomal dominant disorder characterized by myopathy and progressive muscle weakening in the shoulder girdle and peroneal muscles. The disease has late onset, with affected individuals first displaying symptoms in their late teens or early twenties and up to the late fifties. This disease affects the legs and feet (with foot drop and hammer toes) and the proximal and/or distal arms. Patients have scapular winging and asymmetry. There is intolerance to exercise. Other symptoms include contractures, hearing loss twitching muscle cramps, facial weakness and cardiac disorders. Death results from cardiac or respiration failure. Although the underlying genetic defect has not been identified previously, SPMD has been mapped by genetic linkage analysis to human chromosome 12q13.3-q15.
The defective association of skeletal and cardiac muscle with their surrounding basal lamina underlies the pathologies associated with a variety of muscular dystrophies and cardiomyopathies (Matsumura and Campbell, 1994, Hayashi, et al., 1993; Hayashi, et al., 1988; Lim and Campbell 1998). Duchenne Muscular Dystrophy (DMD) is a congenital X-linked myopathy that is caused by a lack of the dystrophin protein and affects approximately 1 in 3300 males. Patients with DMD experience progressive muscle deterioration and debilitation that severely restricts mobility. Death due to cardiac and respiratory failure usually occurs in the second decade of life.
Mutations in the dystrophin gene result in a lack of dystrophin, a 427 kDa protein localized to the inner cytoplasmic side of the plasma membrane of skeletal and cardiac muscle cells (Monaco et al., 1986; Matsumura and Campbell, 1994; Campbell, 1995). In association with dystroglycans, syntrophins, and sarcoglycans, dystrophin links the cell cytoskeleton to laminin in the extracellular matrix. In the absence of one or more components of the dystrophin linkage system, the association of fibers with the surrounding basal lamina is compromised, leading to the myopathy observed. Thus, the molecular continuity between the extracellular matrix and the cell cytoskeleton is essential for the structural and functional integrity of muscle.
The integrins are αβ heterodimeric receptors that bind extracellular matrix proteins and interact with the cell cytoskeleton (Hynes, 1992). The α7β1 integrin is a laminin receptor on skeletal and cardiac muscle (Song et al., 1992) and serve as a transmembrane link between the basal larnina and muscle fibers. Multiple insoforms of the α7 and β1 chains are generated by developmentally regulated RNA splicing resulting in a family of receptors with diverse structure and functions (for reviews see Hodges and Kaufman, 1996 and Burkin and Kaufman, 1999).
The α7 integrin chain is encoded by a single autosomal gene on human chromosome 12q13 (Wang et al., 1995). Three alternative cytoplasmic domain (α, 7A, B and C) and two extracellular domain variants (X1 and X2) of the protein have been identified (Song, et al., 1993; Collo et al., 1993, Ziober et al., 1993). Four additional alternatively spliced isoforms of the extracellular domain have been predicted by nucleotide sequence analysis (Leung et al., 1998; Vigner, et al., 1999).
The α7β1 integrin is a major laminin receptor that serves as a transmembrane link and signal transduction mechanism between the extracellular matrix and the muscle fiber (Song et al., 1992, Hodges and Kaufman, 1996; Burkin and Kaufman, 1999). Alternative cytoplasmic domains (A, B and C) Song et al. 1993; Collo et al., 1993; Zoiber et al. 1993) and extracellular domains (X1 and X2) (Zoiber et al., 1993, Hodges and Kaufman, 1996) of this integrin are generated by developmentally regulated alternative RNA splicing. The diversity in the α7 integrin chain appears to be the result of the broad range of biological functions with which it is associated during muscle development, including the development of neuromuscular junctions (Burkin et al., 1998; Burkin et al., 2000), stability of myotendinous junctions and overall muscle integrity (Hayashi et al., 1998).
The β1 chain cytoplasmic domain also undergoes developmentally regulated alternative splicing β1A is the most common isoform of the β1 chain and is expressed in a wide variety of tissues including replicating myoblasts. The alternative β1D form is generated upon differentiation of myoblasts to myofibers (Zhidkova et al., 1995; van der Flier et al., 1995, Belkin et al., 1996; Belkin et al., 1997).
Mutations in the genes that encode the many components of the dystrophin glycoprotein complex cause the majority of muscular dystrophies. Mutations in the α7 gene also cause congenital myopathics (Hayashi et al., 1998). Thus, both the integrin and dystrophin-mediated transmembrane linkage systems contribute to the functional integrity of skeletal muscle. Interestingly, there is an increase in the amount of α7 transcript and protein in DMD patients and mdx mice (the mouse model that has a mutation in its dystrophin gene) (Hodges et al., 1997). This led us to suggest that enhanced expression of the integrin may partially compensate for the absence of the dystrophin glycoprotein complex (Hodges, et al., 1997; Burkin and Kaufman, 1999). Utrophin, a protein homologous to dystrophin, is also increased in DMD patients and mdx mice (Law, et al., 1994). Utrophin associates with many of the same proteins as dystrophin, and further increasing utrophin may, in part, also compensate for the absence of dystrophin (Tinsley et al., 1996).
Although DMD patients (Monaco et al., 1987) and mdx mice (Bulfield et al., 1984; Sicinski, 1989) both lack distrophin, the pathology that develops in the mdx mouse is much less severe than that observed in humans. The differences in the extent of pathology may be due to a number of factors including the enhanced expression and altered localization of utrophin (Law; et al., 1994; Pons et al., 1994) and the α7 integrin chain (Hodges et al., 1997) in mdx mice. In addition, differences in utilization of skeletal muscles by humans compared to mice in captivity may also contribute to the decreased level of pathology seen in mdx mice. In contrast, mdx/utr (−/−) mice lack both of dystrophin and utrophin and have a phenotype that is similar to that seen in Duchenne patients. These double mutant mice develop severe progressive muscular dystrophy and die prematurely between 4-20 weeks of age (Grady et al. 1997b, Deconinck, et al., 1997b).
To explore the hypothesis that enhanced expression of the α7β1 integrin compensates for the absence of the dystrophin glycoprotein complex and reduces the development of severe muscle disease, transgenic mice were made that express that rat α7 chain. The mdx/utr (−/−) mice with enhanced expression of the α7BX2 chain isoform show greatly improved longevity and mobility compared to non-transgenic mdx/utr (−/−) mice. Transgenic mice maintained weight and had reduced spinal curvature (kyphosis) and joint contractures. Transgenic expression of the α7BX2 chain also reduced the degree of mononuclear cell infiltration and expression of fetal myosin heavy chain (fMyHC) in muscle fibers. Together these results show that enhanced expression of α7BX2β1D integrin significantly reduces the development of muscular dystrophy.
Muscle fibers attach to laminin in the basal lamina using the dystrophin glycoprotein complex and the α7β1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy, β2 laminin congenital muscular dystrophy, sarcoglycan related muscular dystrophy, and α7 integrin congenital muscular dystrophy. Therefore the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the α7β1 integrin can compensate for the absence of dystrophin, we have expressed the rat α7 chain in mdx/utr (−/−) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that observed in Duchenne muscular dystrophy, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the α7BX2 integrin chain was increased approximately 2 3-fold in mdx/utr (−/−) mice. Concomitant with the increase in the α7 chain, its heterodimeric partner, β1D, was also increased in the transgenic animals. The transgene expression of the α7BX2 chain in the mdx/utr (−/−) mice extended their longevity by three-fold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuronmuscular junction. Thus bolstering α7β1 integrin mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr (−/−) mice and compensates for the absence of the dystrophin- and utrophin- mediated linkage systems.
There is a longfelt need in the art for definitive and accurate methods for the diagnosis of particular types of neuromuscular disorders, such as SPMD, and to characterize the particular defects of the disorder. Direct or indirect (e.g. drug) treatment is likewise unavailable, through need. Enhanced expression of the α7β1 integrin provides a novel approach for and fulfills a longfelt need for treatment of Duchenne muscular dystrophy and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex.